Nucleic acid polymer/intercalator monolayer and methods of using and making thereof

ABSTRACT

The present invention has an object to provide an easy method for detecting a nucleic acid polymer in aqueous phase. 
     The present invention provides a method for detecting the amount of nucleic acid polymer, which comprises the steps of modifying an intercalator to be amphiphilic by using a hydrophobic group, spreading the amphiphilic intercalator on an aqueous solution containing a nucleic acid polymer to form a monolayer of said nucleic acid polymer and said amphiphilic intercalator at the gas-water interface, and measuring surface pressures per unit area of said monolayer.

This is a divisional application of Ser. No. 08/595,206 filed Feb. 1,1996 now U.S. Pat. No. 5,871,915.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for detecting a nucleic acidpolymer. More particularly, the present invention relates to a methodwhich permits easy detection of an amount of a nucleic acid polymer in asample, detection of hybridization, if any, of a probe and a nucleicacid polymer, identification of a base sequence of a nucleic acidpolymer, a method for causing two-dimensional orientation of a nucleicacid polymer at the gas-water interface, and further, a novelamphiphilic intercalator used in these methods.

2. Description of Related Art

Diverse and various biological functions observed in cells areeffectively expressed by regular orientation of biomolecules. Fornucleic acid polymers (DNA, RNA) which code genetic information of anorganism, however, the effect of an orientation thereof on expression ofbiological functions has almost never been studied. One of the reasonsis that means to control in vitro the orientation of a nucleic acidpolymer has not as yet been established.

As a method for retrieving a target gene sequence in a nucleic acidpolymer, or for determining similarities and differences or homology ofa plurality of nucleic acid polymers, on the other hand, it isconventionally known that the hybridization method using, as a probe, asingle-stranded nucleic acid polymer (DNA or RNA) complementary with aportion of sequence of a target nucleic acid polymer. More specifically,the conventional hybridization method comprises the steps of fixing asingle-stranded target nucleic acid polymer onto a nitrocellulosemembrane or a nylon membrane, and adding an aqueous solution of a probenucleic acid polymer labelled with a radioisotope or an enzyme onto themembrane. When the probe nucleic acid polymer is hybridized with thetarget nucleic acid polymer, only the hybridized probe nucleic acidpolymer remains on the membrane after washing. Presence of a searchedsequence in the target nucleic acid can be determined by detectingradioactivity from the radioisotope labelled on the probe nucleic acidpolymer, or chemiluminescence or color of precipitate produced by theenzyme.

In order to handle a radioisotope, however, it is necessary to acquire aspecial license, so that this technique is not popularly accepted.Labelling a single-stranded probe nucleic acid polymer with an enzymerequires much costs and labor.

A nucleic acid polymer such as genomic DNA existent in chromosome has adouble helix structure comprising complementary base pairs(adenine/thymine and cytosine/guanine for DNA, and adenine/uridine,cytosine/inosine and cytosine/guanine for RNA). For identifyingdifferences in the base sequence between two different nucleic acidpolymers, for example, formation of a triple helix has been believed tobe effective. However, because of the difficulty to detect formation ofa triple helix, this method has not as yet been put to practical use.

Among properties of DNA or RNA as a nucleic acid polymer, there is knownan intercalation phenomenon in which a cationic pigment is insertedbetween neighboring base pairs. However, detection of a nucleic acidpolymer (content, hybridization, identification of base sequence, etc.)by the use of this phenomenon has not as yet been conducted.

SUMMARY OF THE INVENTION

The present invention has as an object to provide a method for detectingan amount of a nucleic acid polymer in an aqueous solution and thepresence of hybridization of nucleic acid polymer/probe by theutilization of interaction between a pigment (intercalator) and thenucleic acid polymer, and a method for identifying the base sequence ofthe nucleic acid polymer.

More specifically, the first invention provided by the present inventionis a method for detecting an amount of nucleic acid polymer, whichcomprises the steps of modifying an intercalator to be amphiphilic byusing a hydrophobic group, spreading the amphiphilic intercalator on anaqueous solution containing a nucleic acid polymer to form a monolayerof said nucleic acid polymer and said amphiphilic intercalator, andmeasuring surface pressure per unit area of said monolayer at thegas-water interface.

The second invention relates to a method for detecting the presence ofhybridization of a probe nucleic acid polymer and a target nucleic acidpolymer, which comprises the steps of modifying an intercalator to beamphiphilic by using a hydrophobic group, spreading the amphiphilicintercalator on an aqueous solution containing a single-stranded probenucleic acid polymer to form a monolayer of said probe nucleic acidpolymer and said amphiphilic intercalator at the gas-water interface,measuring a surface pressure-area isotherm of said monolayer, thenmeasuring a surface pressure-area isotherm of said monolayer afteraddition of a single-stranded target nucleic acid polymer to the aqueoussolution, and comparing the two surface pressure-area isotherms.

The third invention relates to a method for identifying a base sequenceof a nucleic acid polymer, which comprises the steps of modifying anintercalator to be amphiphilic by using a hydrophobic group, spreadingthe amphiphilic intercalator on an aqueous solution containing a nucleicacid polymer to form a monolayer of the nucleic acid polymer and theamphiphilic intercalator at the gas-water interface, and measuringsurface pressure-area isotherm of said monolayer.

The present invention has another object to provide a method for causingtwo-dimensional orientation of a nucleic acid polymer at the gas-waterinterface.

More specifically, the fourth invention is a method for orientatingnucleic acid polymers at the gas-water interface, which comprises thestep of modifying an intercalator to be amphiphilic by using ahydrophobic group, spreading the amphiphilic intercalator on an aqueoussolution containing nucleic acid polymers to form a monolayer of saidnucleic acid polymers and said amphiphilic intercalator.

Furthermore, the present invention provides an intercalator modified tobe amphiphilic by using a hydrophobic group and a nucleic acidpolymer/intercalator monolayer comprising this intercalator and thenucleic acid polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation illustrating a method for detectingthe presence of hybridization in the present invention;

FIG. 2 is a schematic representation illustrating a method foridentifying a base sequence in the present invention;

FIG. 3 is an NMR spectrum of an intercalator (Formula 1) of the presentinvention and FIG. 4 is an IR spectrum thereof;

FIG. 5 illustrates the difference in surface pressure-area isothermsbetween the presence and absence of hybridization;

FIG. 6 is an NMR spectrum of another intercalator (Formula 2) of thepresent invention;

FIG. 7 illustrates the difference in surface pressure-area isothermsbetween different base sequences;

FIG. 8 illustrates changes in the difference in surface pressuredepending upon concentrations of a nucleic acid polymer; and

FIG. 9 is an atomic force microscopic image of an intercalator/DNAmonolayer deposited on a mica substrate.

FIG. 10 is a schematic drawing of the microscopic image of FIG. 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention permits detection of a nucleic acid polymer byutilizing intercalation of a pigment, forming a monolayer of anintercalator and a nucleic acid polymer at the gas-water interface, andmeasuring a surface pressure of this monolayer. The method ischaracterized in that a surface active pigment modified by a hydrophobicgroup into an amphiphilic one is used as the intercalator. Theindividual detection methods are described below in detail.

<A> Detection of Nucleic Acid Polymer Amount:

First, a nucleic acid polymer (single-stranded or double-stranded DNA orRNA) is added into an aqueous subphase of an ordinary surfacepressure-area isotherm measuring apparatus. A monolayer is formed byspreading the surface-active intercalator of the present invention atthe gas-water interface. More specifically, since this intercalator hasa positive charge, it forms a polyion complex with the nucleic acidpolymer having a negative charge at the gas-water interface, thusforming a monolayer. The surface pressure of this monolayer varies,depending upon the amount of nucleic acid polymer coupled with theintercalator. By measuring the surface pressure thereof per unit area(area occupied by the monolayer), therefore, it is possible to detectthe amount of the nucleic acid polymer in the aqueous solution. Moreparticularly, the amount can be detected by determining the differencebetween the measured surface pressure and the surface pressure of purewater not containing a nucleic acid polymer. By previously preparing acalibration curve of differences in surface pressure by the use ofnucleic acid polymer aqueous solutions having various knownconcentrations, furthermore, it is possible to easily detect the amountof a nucleic acid polymer of an unknown concentration.

<B> Hybridization:

First, as shown in FIG. 1, a single-stranded probe nucleic acid polymer(DNA or RNA) is added into an aqueous subphase of an ordinary surfacepressure-area isotherm measuring apparatus. A monolayer is formed byspreading the surface-active intercalator solution onto the gas-waterinterface thereof. At this point, as the intercalator has a positivecharge, it forms a polyion complex with the single-stranded nucleic acidpolymer having a negative charge at the gas-water interface, thusforming a monolayer. The surface pressure-area isotherm (π-A isotherm)of the thus formed monolayer is measured. Then a single-stranded nucleicacid polymer which may have a target sequence to be detected is added tothe aqueous subphase. When the target nucleic acid polymer has a basesequence complementary with the probe, the single-stranded probe nucleicacid polymer having formed the polyion complex with the intercalator ishybridized with the single-stranded target nucleic acid polymer, thusforming a polyion complex comprising the intercalator anddouble-stranded probe/target nucleic acid polymer at the gas-waterinterface. Because the pigment portion of the intercalator is insertedbetween base pairs of the double-stranded probe/target nucleic acidpolymer, there is created a surface pressure-area isotherm differentfrom that before hybridization. This difference in the surfacepressure-area isotherm makes it possible to detect the presence ofhybridization.

<C> Identification of Difference Between Base Sequences of Nucleic AcidPolymer:

As shown in FIG. 2, for example, an aqueous solution of a target doublehelix nucleic acid polymer (DNA or RNA) is added into the aqueoussubphase of an ordinary surface pressure-area isotherm measuringapparatus. A monolayer is formed by spreading a surface activeintercalator solution at the gas-water interface thereof. At this point,since the surface-active intercalator has a positive charge, it forms apolyion complex at the gas-water interface with the nucleic acid polymerhaving a negative charge, thus forming a monolayer. Further, the pigmentintercalator portion intercalates with the double helix nucleic acidpolymer. The surface pressure-area isotherm thereof is measured. Becausethe surface pressure-area isotherm largely depends upon the basesequence of the double helix nucleic acid polymer, it is possible toidentify, from the surface pressure-area isotherm, the kind of basesequence of a nucleic acid polymer existent in the aqueous subphase.That is, the base sequence can be identified by previously preparingsurface pressure-area isotherms for nucleic acid polymers having variousknown sequences, and comparing a tested sequence with these isotherms.

<D> Method for Causing Two-dimensional Orientation of Nucleic AcidPolymer:

First, an aqueous solution of a nucleic acid polymer (single-stranded ordouble-stranded DNA or RNA) is added into an aqueous subphase of anordinary surface pressure-area isotherm measuring apparatus. A monolayeris formed by spreading a solution of the surface-active intercalator ofthe present invention at the gas-water interface thereof. Because thisintercalator has a positive charge, it forms a polyion complex at thegas-water interface with the nucleic acid polymer having a negativecharge, thus forming a monolayer. By compressing or dispersing thismonolayer, for example, while controlling the surface pressure of themonolayer, it is possible to control orientation of the nucleic acidpolymer coupled with the intercalator.

Now, the surface-active pigment intercalator used in the method of thepresent invention will be described in detail below.

Intercalation is observed in pigments such as acridine orange andethidium bromide. In the present invention which utilizes formation of acomplex with a nucleic acid, any of these various pigment intercalatorsincluding these conventional ones is modified by a hydrophobic group toimpart an amphiphilic surface activity.

A typical hydrophobic group used here is alkyl group. The presentinvention proposes, as a more preferable one, a compound modified byC_(n) H_(2n+1) (n≧13) alkyl group. For example, surface-activeintercalators of the following formulae, available by modifying acridineorange with octadecyl group are provided: ##STR1##

The compounds expressed by Formulae 1 and 2 are surface-activeintercalators so far unknown, which can easily be synthesized byreacting an octadecyl iodine or a derivative thereof with the compoundskeleton of acridine orange. The compound of Formula 1 is asurface-active intercalator having one hydrophobic chain, and thecompound of Formula 1 is a surface-active intercalator having twohydrophobic chains.

These compounds will be described below by means of Examples of thepresent invention.

EXAMPLE 1

The presence of hybridization was measured with the use of the compoundof Formula 1 above. The compound had the following properties:

(1) recrystallization from benzene:rubiginous imbricate crystal;

(2) melting point: 202.5˜204.5° C.;

(3) TLC:Rf=0.5 (chloroform/methanol=9/1+some drops of acetic acid).

In addition, the elemental analysis of this compound is that of Table 1.

                  TABLE 1                                                         ______________________________________                                                       C      H         N    I                                        ______________________________________                                        Theoretical Value (%)                                                                        65.10  8.74      6.51 19.65                                      Analytical Value (%) 63.81 8.47 6.70 21.26                                  ______________________________________                                    

MHR and IR data are shown in FIGS. 3 and 4.

The results of actual measurement of surface pressure-area isotherms forthis compound are shown in FIG. 5. The subphase exchange type filmbalance controlled by a microprocessor (made by USI Systems Company) wasemployed for measurement of surface pressure-area isotherms. With atrough area of 220×100 mm², the surface pressure was measured with theuse of filter paper (1 cm×1 cm) by the Wilhelmy method. While measuringsurface pressure-area isotherms, temperature of the aqueous subphase waskept constant (20° C. by means of a circulator. A chloroform (specialclass) solution of a surface-active intercalator (10 mg/10 ml) in anamount of 15 μl was spreaded on the aqueous subphase containing anucleic acid polymer to measure surface pressure-area isotherms at acompression rate of 0.04 nm² /min/molecule.

In addition, the following conditions were adopted:

Polyadenylic acid concentration of aqueous subphase:

10 mg/1000 ml (pure water), pH: 5.6

Inverted polyuridylic acid concentration:

10 mg/1000 ml (pure water), pH: 5.6.

As a model of single-stranded nucleic acid polymer, polyadenine wasused, and as a model of single-stranded target nucleic acid polymer,complementary polyuridine was employed. A large change in surfacepressure-area isotherms was confirmed by the addition of polyuridine tothe aqueous subphase.

EXAMPLE 2

A base sequence of DNA was identified by the use of the compound ofFormula 2 above. The compound was purified from silica gel column byusing a solution of chloroform/methanol (=95/5) as an eluate, andTLC:Rf=0.5 (chloroform/methanol=9/1+some drops of acetic acid).

NMR data are shown in FIG. 6.

The results of actual measurement of surface pressure-area isotherms forthis compound are shown in FIG. 7.

Measurement was carried out in the same manner as in Example 1, withother conditions including a concentration of the double helix nucleicacid polymer in the aqueous subphase of 10 mg/1000 ml (pure water) and apH of 5.6. The graph is a surface pressure-area isotherm for the casewhere polyadenylic acid-polyuridylic acid and polyinosinicacid-polycytidylic acid were present in the aqueous subphase as modelsof double helix nucleic acid polymer.

More specifically, as shown in FIG. 7, the base sequence can beidentified from a change in the surface pressure by adding a doublehelix nucleic acid polymer to the aqueous subphase if the gas-waterinterface has a constant surface area.

EXAMPLE 3

An aqueous solution was prepared by dissolving a double helix DNA(extracted from salmon spermatozoon) in pure water, to a DNAconcentration within a range of from 0.01 to 100 mg/1000 ml and a pH of5.6. A chloroform solution of the surface-active intercalator of Formula1 was spreaded onto the gas-water interface of this aqueous solution,and the surface pressure-area isotherm was measured by the same methodunder the same conditions as in Example 1. The surface pressure wasmeasured with a molecule-occupying area of 0.8 nm² /molecule, and on theother hand, pressure on the pure water surface with the same area wasmeasured to determine a difference between them for each value of DNAconcentration, thus preparing a calibration curve as shown in FIG. 8.

As is clear from FIG. 8, the difference in surface pressure wasconfirmed to exhibit a correlation with logarithm of DNA concentrationwithin a range of DNA concentration of from 0.1 to 10 mg/1000 ml.

EXAMPLE 4

An aqueous solution was prepared by dissolving a double helix DNA(extracted from salmon spermatozoon) in pure water, to a DNAconcentration of 10 mg/1000 ml and a pH of 5.6. A chloroform solution ofthe surface-active intercalator of Formula 1 was spreaded onto thegas-water interface of this aqueous solution, and the interface wascompressed while measuring the surface pressure by the same method underthe same conditions as in Example 1. The compression was discontinuedwhen the surface pressure reached 10 mN/m, and control was carried outso as to always keep this value of the surface pressure. A freshlycleaved mica substrate had previously been immersed vertically in theaqueous subphase of the apparatus. A single layer of a compositemonolayer comprising the intercalator and the DNA was derposited on thesubstrate by pulling up this substrate vertically at a speed of 50mm/minute. After air drying, the substrate surface was observed in ACmode (scanning area: 200×200 nm²) by the use of an atomic forcemicroscope (NV2500, made by Olympus). The result is as shown in themicroscopic image of FIG. 9 and the shematic drawing thereof of FIG. 10.The monolayer is compressed from right toward left in this image. Cordshaving a width of from 10 to 20 nm were observed in parallel sequencewith a difference of 2 to 3 Å, and DNA molecules were found to bevertically oriented relative to the compression direction in bundles. Itwas confirmed from these results that it was possible to controltwo-dimensional orientation of a nucleic acid polymer by combinationwith the intercalator of the present invention. In addition, thusobtained nucleic acid polymer/intercalator monolayer or the monolayerdeposited on a substrate, in which polymer molecules or bundles of themolecules are oriented in the same direction. will be used for amolecule devise such as micro-conductor, sensor and a tool for studyingthe orientation state of DNA or RNA.

Particulally, for example, Br-- which is a conventional intercalator iswater-soluble because of a short hydrophobic group as C₁₂ H₂₅, so thatdevelopment thereof on a gas-liquid interface only causes dissolutioninto the aqueous subphase, a no rise of surface pressure is observedeven by compression. In the methods of the present invention describedin the above-mentioned examples, therefore, it is essential to use, nota conventional one, but a surface-active intercalator modified by ahydrophobic group into an amphiphilic one.

What is claimed is:
 1. A method for orienting nucleic acid polymers,which comprises spreading an amphiphilic intercalator containing ahydrophobic group on an aqueous solution containing nucleic acidpolymers to intercalate said amphiphilic intercalator into said nucleicacid polymers, thereby orienting said nucleic acid polymers at agas-water interface of the aqueous solution.
 2. A nucleic acidpolymer/intercalator monolayer comprising a monolayer of nucleic acidpolymers which are intercalated with an amphiphilic intercalatorcontaining a hydrophobic group, said hydrophobic group being an alkylgroup expressed by C_(n) H_(2n+1) (n≧13).
 3. The nucleic acidpolymer/intercalator monolayer of claim 2, which exists at a gas-waterinterface of an aqueous solution.
 4. A nucleic acid polymer/intercalatormonolayer of claim 2, which exists on a substrate.
 5. The nucleic acidpolymer/intercalator monolayer of claim 2, wherein the intercalator isacridine orange.
 6. The nucleic acid polymer/intercalator monolayer ofclaim 2, wherein the hydrophobic group is an octadecyl group.
 7. Thenucleic acid polymer/intercalator monolayer of claim 2, wherein theamphiphilic intercalator containing the hydrophobic group is expressedby any one of the following formulae: ##STR2##
 8. A nucleic acidpolymer/intercalator monolayer obtained by a method comprising:spreading an amphiphilic intercalator containing a hydrophobic group onan aqueous solution having a gas-water interface and containing nucleicacid polymers to intercalate said amphiphilic intercalator into saidnucleic acid polymers and form a monolayer of said nucleic acid polymersand said amphiphilic intercalator at the gas-water interface,andisolating the monolayer from the gas-water interface of the aqueoussolution to obtain the nucleic acid polymer/intercalator monolayer. 9.The nucleic acid polymer/intercalator monolayer of claim 8, which isdeposited on a substrate after surface compression.
 10. The nucleic acidpolymer/intercalator monolayer of claim 8, wherein the hydrophobic groupis an alkyl group expressed by C_(n) H_(2n+1) (n≧13).
 11. The nucleicacid polymer/intercalator monolayer of claim 8, wherein the intercalatoris acridine orange.
 12. The nucleic acid polymer/intercalator monolayerof claim 8, wherein the hydrophobic group is an octadecyl group.
 13. Thenucleic acid polymer/intercalator monolayer of claim 8, wherein theamphiphilic intercalator containing the hydrophobic group is expressedby any one of the following formulae: ##STR3##
 14. A method of making anucleic acid polymer/intercalator monolayer comprising: spreading anamphiphilic intercalator containing a hydrophobic group on an aqueoussolution having a gas-water interface and containing nucleic acidpolymers to intercalate said amphiphilic intercalator into said nucleicacid polymers and form a monolayer of said nucleic acid polymers andsaid amphiphilic intercalator at the gas-water interface, andisolatingthe monolayer from the gas-water interface of the aqueous solution tomake the nucleic acid polymer/intercalator monolayer.
 15. The method ofmaking a nucleic acid polymer/intercalator monolayer of claim 14,wherein the hydrophobic group is an alkyl group expressed by C_(n)H_(2n+1) (n≧13).
 16. The method of making a nucleic acidpolymer/intercalator monolayer of claim 14, wherein the intercalator isacridine orange.
 17. The method of making a nucleic acidpolymer/intercalator monolayer of claim 14, wherein the hydrophobicgroup is an octadecyl group.
 18. The method of making a nucleic acidpolymer/intercalator monolayer of claim 14, wherein the amphiphilicintercalator containing the hydrophobic group is expressed by any one ofthe following formulae: ##STR4##